This study investigated plasma lipopolysaccharides (LPS) concentration and intestinal permeability after 60-min run at 70 % maximum oxygen uptake (VO2max) in hot [33 °C, 50 % relative humidity (rH)] and cool (22 °C, 62 % rH) conditions. Fifteen volunteers gave their informed consent to participate in this study. Their venous blood samples were taken before, after, 2 and 5 h after exercise in each of the conditions. The order of the two environmental conditions in which they exercised in was randomised and counterbalanced. Plasma LPS concentration increased by an average of 54.0 % (95 % confidence interval: 30.7, 75.1 %) after exercising in the hot trial but no significant changes were observed in cool trial, where mean plasma LPS concentration was 12.0 ± 6.4 pg mL(-1) (before), 10.9 ± 5.4 pg mL(-1) (after), 10.7 ± 6.0 pg mL(-1) (2 h after) and 10.6 ± 5.7 pg mL(-1) (5 h after). Median (range) plasma claudin-3 (CLDN3) concentration was significantly higher after exercise (hot: 8.2 [1.0-13.0] ng mL(-1) and cool: 7.6 [0.6-13.4] ng mL(-1)) as compared to before exercise (hot: 6.6 [0.7-11.8] ng mL(-1) and cool: 6.7 [0.8-12.6] ng mL(-1)) (p < 0.05), but there is no significant difference observed between trials (p > 0.05). Changes in intestinal permeability are only affected by exercise while exercise-induced endotoxemia is affected by environmental conditions. This study, thus, highlights that an increase in intestinal permeability is not sufficient to trigger exercise-induced endotoxemia, suggesting that post-LPS translocation events may have a greater impact in its occurrence.